1. Field of the Invention
The present invention relates to a recombinant microorganism having improved putrescine productivity and a method for producing putrescine at a high yield using the same.
2. Description of the Related Art
Polyamines such as spermidine, spermine or the like are present in most living cells, and putrescine (or 1,4-butanediamine) is used as a precursor in spermidine and spermine metabolisms. Putrescine is found in Gram-negative bacteria or fungus, and it is present in high concentrations in various species, suggesting that it has an important role in the metabolic pathways of microorganisms.
In general, putrescine is an important raw material in a synthesis of polyamine nylon-4, 6 which is produced by reacting with adipic acid. Putrescine is produced mainly by chemical synthesis through acrylonitrile and succinonitrile from propylene. This chemical synthesis is a three-step process including a catalytic oxidation reaction, a reaction using a cyanide compound, and a hydrogenation reaction using high-pressure hydrogen. There are problems in that this chemical synthesis is not environment friendly and also consumes a lot of energy leading to depletion of petroleum. Therefore, a more environment friendly and energy-effective method involving biomass utilization needs to be developed for putrescine production.
In microorganisms, a biosynthetic pathway of putrescine is the same as route of arginine synthesis from glutamate to ornithine synthesis. Putrescine can be biosynthesized through two pathways from microorganisms. In one pathway, ornithine as an intermediate is decarboxylated to synthesize putrescine. In the other pathway, agmatine is produced by decarboxylation arginine synthesized from ornithine, and then putrescine is synthesized from the agmatine (Morris et al., J Biol. Chem. 241: 13, 3129-3135, 1996). These two pathways produce the energy required for metabolism or allow the cell to have resistance to oxidative stress.
As a method for producing putrescine using a microorganism, a method for producing putrescine at a high concentration by transformation of E. coli and Corynebacterium has been reported (International Patent Publication No. WO06/005603; International Patent Publication No. WO09/125924; Qian ZD et al., Biotechnol. Bioeng. 104: 4, 651-662, 2009; Schneider et al., Appl. Microbiol. Biotechnol. 88: 4, 859-868, 2010; Schneider et al., Appl. Microbiol. Biotechnol. 91: 17-30, 2011). For example, WO09/125924 discloses a method for producing putrescine in a high yield by enhancing ornithine biosynthetic pathway, instead of inactivating pathways involved in degradation and utilization of putrescine which are present in E. coli and inactivating conversion of ornithine as a precursor of putrescine to arginine. In addition, Schneider (2010) discloses a method for producing putrescine at a high concentration by introducing and enhancing a protein capable of converting ornithine to putrescine into a Corynebacterium sp. strain having no putrescine productivity.
Furthermore, studies on putrescine transporters in E. coli, yeast, plant and animal cells have been actively conducted (K Igarashi, Plant Physiol. Biochem. 48: 506-512, 2010). Putrescine uptake of E. coli occurs via 4 pathways; potABCD or potFGHI driven by ATP hydrolysis, andpotE as H+ symporter and puuP of the puu pathway. With regard to Km values of these complexes involved in putrescine uptake, those of PotFGHI, potABCD, potE and puuP are 0.5 mM, 1.5 mM, 1.8 mM, and 3.7 mM, respectively. Among the four putrescine uptake pathways, potFGHI complex is considered as the most suitable. In addition, potE transporter has both functions of uptake and excretion of putrescine. Putrescine is imported together with proton into cells at neural pH. However, as putrescine synthase (speF) is expressed under acidic pH conditions, intracellular uptake of extracellular ornithine and extracellular excretion of putrescine synthesized within cells occur at the same time (Kurihara et. al., J. Bacteriology 191: 8, 2776-2782, 2009).
The known putrescine exporters in yeast are TPO1 and TPO4. These amino acid sequence are very similar to the amino acid sequence of bacillus multidrug transporter Blt.
These two exporters share characteristics with potE in E. coli, and they have functions of importing putrescine, spermidine, and spermine under basic conditions and exporting them under acidic conditions. In addition, yeast cell over-expressing TPO5 gene is resistant to 120 mM putrescine whereas a mutant disrupted TPO5 gene is sensitive to 90 mM putrescine (Tachihara et. al., J. Biological Chemistry, 280(13): 12637-12642, 2005).
Synthesis and degradation, and uptake and excretion of putrescine in animal cells are regulated in various ways. Although studies on polyamine excretion have not been done in animal cells as well as in E. coli or yeast, there is a report that an SLC3A2 (arginine/diamine exporter) functions to import arginine into cells and to export putrescine, acetyl spermidine, and acetyl spermine in colon epithelial cells. However, there has been no report about uptake and export of putrescine in plant cells (Igarashi et al., Plant Physiol. & Biochem. 48: 506-512, 2010).
On the other hand, since Corynebacterium sp. microorganism has no putrescine biosynthetic pathway, studies regarding putrescine export have not been studied. According to a recent report, cell growth is restored and cadaverine productivity is increased by overexpression of a cg2983 membrane protein in a strain producing a cadaverine (Kind et. al., Metabolic Engineering 13: 617-627, 2011).
However, there have been no reports about association between putrescine exporter and putrescine productivity or growth of microorganisms producing putrescine. In the above literature, there is no mention about association between cg2983 membrane protein and the exporting ability of putrescine.
In this background, the present inventors have made many efforts to develop a strain capable of producing putrescine in a higher yield. As a result, NCgl2522 functions is revealed as a putrescine exporter in a putrescine-producing strain, Corynebacterium sp. microorganism, and putrescine can be produced in a high yield by enhancing NCgl2522 activity, compared to the endogenous activity thereof. In addition, the amount of putrescine in a culture medium can be increased by expressing NCgl2522 in E. coli having the putrescine synthetic pathway, and thus the present inventors suggested that NCgl2522 also functions as a putrescine exporter in E. coli, thereby completing the present invention.